AIDS is the fourth leading cause of death globally and, despite antiretroviral combination therapy to delay disease progression and improve quality of life, about 8000 people die of AIDS-related conditions daily. Growing incidences of multiple resistance (and cross-resistance) against approved drugs within patients undergoing highly active antiretroviral therapy (HAART) necessitate development of novel drug to increase the treatment options for HIV/AIDS and to slow further expansion of the epidemic. The long-term objective of this study is to test and develop HIV-1 Nef as a fully validated drug target to increase therapeutic options for HIV carriers, to combat HAART-related drug resistance problems and to improve the quality of life of HIV-1 infected patients. Nef is a unique retroviral protein that is known to be a key determinant of HIV pathogenicity and disease progression. Given the unique and multi-functional properties of Nef, this small protein has a huge potential as a drug target as fortuitous deletions in the nef gene in HIV infected patients of the Sydney Blood Bank Cohort remain essentially free of AIDS related symptoms. These phenotypes have been corroborated by studies in hu-SCID mice and transgenic mouse models for HIV/AIDS. We have established a Nef/N- myristoyl transferase co-expression system and are able to purify large quantities of full-length, wild type, and mutant Nefs in both their myristoylated and unmyristoylated forms. Using solution scattering we have found that the two different forms of the native protein show substantial differences in oligomeric structure and in conformational states. By using a highly interdisciplinary approach, which includes site-directed mutagenesis, fluorescence spectroscopy, solution scattering and single crystal X-ray diffraction methods, we will investigate in detail specific changes in Nef's tertiary and quaternary structure. The specific aims are: ( I ) to determine the high-resolution structures of full-length wild-type and mutant Nef in its myristoylated form using an existing crystal form and ( II ) to develop robust spectroscopic Nef assays with a fast read-out as a platform for future screening campaigns against small molecule inhibitors. The structure of full-length Nef will be used to explore the attachment of fluorescent probes at appropriate and well-defined sites. While results from these experiments will provide us with a more detailed understanding of the structural and functional implications of the post-translational modification, the ultimate goal of this study is to understand the dynamic behavior of Nef in solution in the presence and absence of physiologically relevant host protein ligands. Ultimately, our studies will lay the foundation for developing Nef as a valid drug target. PUBLIC HEALTH RELEVANCE: Globally, HIV/AIDS is the fourth leading cause of death with about 8000 people dying of AIDS-related conditions daily. The overall prevalence of HIV/AIDS, particularly in the developing world, as well as escalating incidences of multi-drug resistance to approved HIV drugs urgently requires the development of novel drug targets such as HIV-1 Nef. Our research is aimed at understanding the dynamic behavior of HIV-1 Nef and establishing this protein as a novel target. Ultimately, we hope to discover potent inhibitors of Nef that will form the basis for novel treatments to cure HIV/AIDS. We expect the eventual outcome of a successful inhibitor screening campaign against HIV-1 Nef, in conjunction with our detailed knowledge of Nef's atomic structure, to be of major medical impact and lead to improvements in the quality of life of HIV-1 infected patients.